A Clinical Trial Shows Improvement in Skin Collagen, Hydration, Elasticity, Wrinkles, Scalp, and Hair Condition following 12-Week Oral Intake of a Supplement Containing Hydrolysed Collagen

Background Hydrolysed collagen supplements are reported to fight the signs of aging and improve skin appearance, but more authoritative clinical evidence is needed to support efficacy. Aim This randomised, double-blind, placebo-controlled study evaluated the efficacy of a supplement containing hydrolysed collagen and vitamin C (Absolute Collagen, AC) on biophysical parameters and visible signs of aging for skin, scalp, and hair, when taken daily or every 48 hours. Methods We measured dermal collagen using confocal microscopy and high-resolution ultrasound. Hydration, elasticity, wrinkles, and clinical trichoscopy were measured in parallel to expert visual grading. Efficacy measures were recorded at baseline, week 6, and week 12. Results Following 12 weeks daily use of the AC supplement, using confocal microscopy, we observed a significant 44.6% decrease in fragmentation vs. placebo (p < 0.01). We also measured a change in the ultrasound LEP (low echogenic pixel) ratio comparing upper and lower dermis (−9.24 vs. −7.83, respectively, p=0.05), suggesting collagen improvements occurred more in the upper dermal compartment. After 12 weeks vs. placebo, skin hydration was increased by 13.8% (p < 0.01), R2 elasticity index was increased by 22.7% (p<0.01), and Rz profilometry index was decreased by 19.6% (p < 0.01). Trichoscopy showed an average 11.0% improvement in scalp scaling and a 27.6% increase in the total number of hairs counted vs. placebo (p=n.s.). This was associated with a 31.9% increase in clinical grading score for hair healthy appearance (p < 0.01). Conclusion The AC supplement has shown clinical benefits for skin, scalp, and hair, when used either daily or every 48 hours, over a 12-week period.


Introduction
Collagen is the most abundant protein in the skin, but its importance runs much deeper than this simple fact.As a protein, collagen is uniquely made for strength (structural rigidity imparted by proline), stability (construction of triple helices), and resilience imparted by extensive crosslinking [1,2].Te formation of dense fbrous networks is pivotal for production of an extracellular matrix scafold with high tensile strength for the skin [3].
Collagen, by way of interaction with elastin and glycosaminoglycan networks, contributes to elasticity, frmness, and skin hydration.Collagen production declines with age and is driven by intrinsic factors (e.g., genetics, hormonal fuxes, and metabolic dysfunction) and extrinsic factors (e.g., UV-radiation, pollution, and smoking) [4].In the aging skin, the ability to replenish collagen naturally decreases by about 1.0% per year [5].Tis decrease in collagen has been linked to the appearance of fne lines and deeper wrinkles.
Stimulation of collagen production and/or inhibition of collagen degradation can be achieved via the use of oral supplements, designed to deliver skin benefts and often referred to as "nutricosmetics."Supplements containing hydrolysed collagen (HC) are reported to fght the signs of the aging process, contributing to a youthful and healthy appearance.When consumed, HC is further hydrolysed to amino acids, dipeptides, and tripeptides.Due to the very high content of hydroxyproline, proline, and glycine in collagen, the resultant peptide species are unique and will not be typically found in other protein sources (e.g., milk and plant-based or fermentation-based vegan equivalents) [6].Tese peptides appear in the bloodstream and skin following ingestion and are ligands proven to stimulate fbroblasts to synthesise new collagen proteins [7,8].
Te HC should be combined with vitamin C (l-ascorbic acid) which, in addition to its role as an antioxidant, can support the synthesis of the collagen triple-helix protein and the assembly of collagen into fbres.Te importance of lascorbic acid is reliant on its use as a cofactor in hydroxylation of proline residues in procollagen (which stabilises the triple-helix structure) and lysine residues (which are used to cross-link fbres) [9,10].As an antioxidant, lascorbic acid can interact with a range of free-radical species to facilitate their neutralisation.Tis is a key mechanism by which the skin is protected from free-radical damage due to UV-radiation and infammaging [11].
Tere have been several reviews of published randomised clinical trials (RCTs) assessing the efcacy of HCbased supplements.A review by Choi et al. carried out a systematic review of 11 studies, with a total of 805 subjects using oral collagen supplementation, and showed signifcant benefts on skin elasticity, hydration, and collagen density [12].Al-Atif summarised the fndings from 12 RCTs that demonstrated improved skin elasticity, turgor, hydration, and reduced skin wrinkling and roughness [13].Pu et al. reported on 26 RCTs assessing the efcacy of the product tested in a total of 1721 subjects and reported benefts of skin hydration and elasticity [14].Te variability of doses, age range, number of weeks tested, and skin measures does little to support the robustness of the clinical reports.However, there is a general pattern suggesting that HC is efcacious as an antiaging treatment for women who use the supplement for up to 12 weeks [15,16].Te conclusion of these systematic reviews and meta-analysis is that HC supplementation can improve skin hydration, elasticity, and wrinkles.
Whilst the clinical benefts of HC for the skin are welldocumented, many of the trials are lacking in a detailed analysis of skin structures such as collagen and therefore do not support the link between the proposed mechanism of collagen production and the putative skin benefts.Tere is also a lack of validated trichological assessments of the scalp and hair, which ideally should be carried out on the same cohort, in the same clinical trial.
Te use of HC supplements for consumers who wish to beneft from improved skin and hair quality, coupled with the proposed ability to fght the signs of aging, has increased rapidly in the last several years.Tere is a need for larger trials with more technical and clinical measures to support this antiaging approach.
We have conducted a clinical trial showing the skin, scalp, and hair benefts of the AC supplement, when used daily, or every 48 hours, and tested against a placebo.To the best of our knowledge, this is the frst report of the efcacy of collagen peptides to improve the skin, scalp, and hair, in the same cohort.For the frst time, we have used high-resolution ultrasound images to show that there are diferential efects following 12 weeks use of the supplement, with increased collagen content in the upper (papillary) dermis compared to the lower (reticular) dermis.Finally, we report the dose regimen is critical to ensure maximal clinical efcacy, with daily use being far superior to use every 48 hours.

Study Design.
Tis study was a randomised, placebocontrolled, double-blind design following the principles of Good Clinical Practice.Te protocol was approved by an independent Ethics Committee (signed by the chairman of the East Anglia Ethics Committee on 09.01.2023).A written declaration of informed consent was received from all participants.Te study conformed to the requirements of the 1964 Declaration of Helsinki.

Subjects.
A total of 140 male and female participants (aged 40-60 years) were enrolled in this study, with 130 participants completing the 12-week trial (Figure 1).Te inclusion criteria included visible signs of aging, requiring a minimum Glogau Wrinkle Score of 2 and a hyperpigmentation score of 4. Fitzpatrick skin types I-VI were recruited, and the split between genders was 90% female and 10% male.Menopausal status was recorded for the female cohort.Exclusion criteria included current skin disease on the face or scalp, allergy or intolerance to test product, aesthetic skin surgery (including Botox), and medications such as anti-infammatory drugs applied directly to the test area (face/scalp).

Test Products.
AC is a skincare supplement containing 8000 mg of hydrolysed collagen, sourced from a sustainable marine supply.Te supplement also contains 60 mg lascorbic acid (75% NRV).A placebo product was manufactured to match the organoleptic properties of the AC supplement, including taste, texture, colour, and viscosity.
For blinding of the clinical trial, both products were presented in a plain white sachet, with only an assigned product code to identify each batch.Te sachets were identical in appearance and labelled according to the ICH-GCP requirements.Subjects were instructed to take 1 sachet per day, or 1 sachet every 48 hours, preferably in the morning, on an empty stomach.Personnel who were not involved in any other aspect of the study were tasked to label the investigational product.Te investigators, statistician, other site personnel, and participants were blinded to the product or placebo.Te unblinding of the clinical trial only occurred after the data were analysed and reported using coded identifers (participant numbers) and coded parallel arm identifers (groups 1-4, representing AC or placebo taken daily and AC or placebo taken every 48 hours).A home use diary was used for compliance checks.Dermatology Research and Practice dermis by dividing the dermal image into two parts of equal thickness.Te ratio of LEP u /LEP i was calculated and allows an assessment of the integrity of the collagen in distinct dermal compartments.

Skin Hydration.
Skin hydration measurements were performed using the Corneometer ® CM825 (Courage and Khazaka, Germany) at baseline, week 6, and week 12. Tis instrument relies on the dielectric constant of water.Any change in the dielectric constant due to skin moisture variations will alter the capacitance of the instrument.Tese variations are detected electronically and are converted into a value represented as arbitrary units.

Elasticity.
Te viscoelastic properties of the skin (elasticity and frmness) were performed using the Cutometer ® MPA 580 (Courage and Khazaka, Germany) at baseline, week 6, and week 12. Te measuring principle is based on the suction method, in which negative pressure is created in the device and the skin is drawn into the aperture of the probe.Te measurements for deformation, passive stretch, and recoil were recorded.For each measurement, a constant suction of 400 mbar was applied for fve seconds, followed by a relaxation time of 3 seconds, measured in triplicate.Elasticity is defned as the skin resistance to mechanical suction force (Ua) vs. its ability to recover after release (Uf ) where R2 � Ua/Uf.

Proflometric Analysis.
A skin surface replica was taken of the crow's feet area of the left side of the face at baseline, week 6, and week 12.A negative replica of the wrinkles on the cutaneous surface of the crow's feet area was obtained using Silfo ® (JS Davis, Hart) material.Proflometric analysis was carried out using the OPTIMAS v6.5 in combination with StatSoft STATISTICA 7. Luminance was measured along a set of 10 equal length parallel lines (passes) running across the replica parallel to the lighting direction.Te variations in luminance were treated as indicative of the roughness and analysed by traditional surface roughness statistics, with Rz reporting the average maximum diference in the luminance value for fve equal length segments in each of the 10 lines traversing the sample.Te ISO defnition of Rz (ten-point height of irregularities) is the average value of the absolute values of the heights of fve highest profle peaks and the depths of fve deepest valleys within the evaluation length.A reduction in the Rz index is indicative of a reduction in fne lines and wrinkles at the crow's feet area.
2.10.Skin Visual Grading.Te Glogau Scale is a validated dermatological tool evaluating skin photoaging, including wrinkles, pigmentation, keratosis, and other skin imperfections.Expert clinical staf applied this 4-point VAS to participants viewed under standardized lighting conditions.Grade 1 corresponds to mild damage to the skin, whereas Grade 4 corresponds to severe photoaging.Expert visual assessors also graded subjects according to evenness of skin tone.A 9-point VAS (where 0 � even skin tone and 9 � very uneven skin tone) was used to follow the putative efects of the AC supplement to improve skin characteristics when compared to a placebo.

Trichoscopy. Te DinoLite Pro Polarising Trichoscope
(Dinolite, the Netherlands) was used to capture images of the hair and scalp.For each subject, 3 areas of the scalp were measured, specifcally the front, mid, and back of the scalp along a central parting to allow capture of high-resolution images.Te images were loaded into analytical software TrichoScience Pro V 1.7 for measurement and data recording.
Te centre section of the scalp was chosen as the measurement area, with measures taken at 4 cm intervals from the hairline to the crown, to assess the scalp condition and the total hair count.Tis was carried out at the start of the trial and repeated at the end of the study at 12 weeks.Te key measurements for the scalp were infammation and scaling, and for hair, we measured the average total hairs per unit area.
Scalp infammation was measured on a 5-point VAS, where a score of 1 represents infammation measured at all 3 test sites and a score of 5 represents normal, healthy, uninfamed scalp.Scalp scaling was measured on a 5-point VAS, where a score of 1 represents scaling that covers more than 30% of all 3 test sites and a score of 5 represents normal, healthy scalp with no visible fakes.Te TrichoScience Pro V1.7 is set to automatically count the total hairs per unit area, and this assessment is carried out for each of the 3 test sites and then averaged.Data are presented as number of hairs per unit area (cm 2 ).
2.12.Hair Visual Grading.Expert visual grading of hair healthy appearance was conducted at baseline, week 6, and week 12. Assessments were made with the aid of a 60-Watt pearl bulb, with distance maintained at approximately 30 cm from the test site.Hair healthy appearance was measured on a 5-point VAS, where a score of 0 represents maximum  [17].
For proflometry Rz, cutometer R2, corneometer, and ultrasound analysis, we used descriptive statistics (mean and standard deviation) and change from baseline (t-test, mean percent change from baseline).For statistical comparison considering treatment vs. placebo, we applied a linear regression analysis (ordinary least squares) model.For the VAS measures including VivaScope 1500, hyperpigmentation, Glogau Scale of wrinkling, photodamage, and hair attributes, we used as above except for the change from baseline (Wilcoxon signed-rank test).For the SPQ responses, percentage of the top 2 positive responses (strongly agree + agree) was reported.Te frequency of "top 2 box" responses was determined for each question.Te signifcance of frequency responses was determined using a chisquared test with a priori 50/50 distribution assumption.

Study Cohorts, Adverse Events, and Protocol Deviations.
A total of 140 volunteers were screened and 138 eligible participants were enrolled, randomised, and split into 4 parallel cohort test groups, comprising treatment vs. placebo, for both use daily or use every 48 hours (Figure 1).Two subjects failed to meet inclusion/exclusion criteria, and a further 8 subjects failed to complete the trial.Tus, the fnal count was 48 subjects taking AC supplement daily vs. 52 taking placebo daily and 14 subjects vs. 16 subjects taking the AC supplement or placebo every 48 hours, respectively.A subset of each group (n � 11 taking the AC supplement daily vs. n � 13 for the other groups) was assigned for clinical trichoscopy.No adverse events were recorded, as expected for a dietary supplement.Two protocol deviations were assessed.A target representation of 20% male participants was not met, as only 10% of male participants who applied and met inclusion criteria were recruited onto the study.One subject was recruited onto the clinical but later an error was found in which the participant was outside the age range for the clinical and was therefore removed from further analysis.Te assessment concluded that these protocol deviations did not signifcantly afect the study.

Te AC Supplement Improves
Collagen Density and Reduces Fragmentation.Expert visual grading of confocal images generated from the VivaScope showed a trend of decreasing collagen fragmentation, corresponding to an improvement in collagen density and structural integrity (Figure 3).As shown in Table 1, following daily use of the AC supplement for 6 weeks, the fragmentation VAS grade had decreased 25.3% compared to the placebo, which showed only minor changes (p < 0.01).By week 12, the fragmentation VAS grade had decreased 44.6% vs. placebo (p < 0.01).Tus, the collagen fbres were increased in density and less fragmented after daily use of AC vs. placebo.

Ultrasound Shows Tat AC Supplement Improves
Collagen Content.By week 12, with daily use of the AC supplement, the HEP signal had increased 231% from baseline (p < 0.01) compared to the placebo which increased 176% from baseline (p < 0.01).Te diference between treatment and placebo was not statistically signifcant.By week 6, the LEP u /LEP i ratio had decreased 16.3% from baseline, compared to a 9.5% decrease for the placebo group (p < 0.01).By week 12, the LEP u /LEP i ratio had decreased 9.3% from baseline compared to a 7.8% decrease for the placebo (p � 0.05).

Use of AC Supplement Improves Hydration,
Elasticity, and Wrinkles.As summarised in Table 1 and Figure 4, using the AC supplement daily, at week 6, the hydration had signifcantly increased 7.5% vs. placebo (p < 0.01).By week 12, a further increase in hydration to 13.8% was measured compared to placebo (which decreased 14.4%, p<0.01).Elasticity was measured as the skin resistance to the mechanical suction force (Ua) versus its ability to recover after release (Uf ), where R2 � Ua/Uf.By week 6, for subjects taking the AC supplement daily, R2 signifcantly increased 20.6% vs. placebo (p < 0.01).By week 12, the R2 had further increased 22.7% vs. placebo (p < 0.01).For measurement of fne lines and wrinkles, the Rz parameter of the proflometry method was used, as this is the accepted clinical standard by which the impact of HC supplement use is measured.By week 6, for subjects taking the AC supplement, the Rz index had decreased 7.7% vs. placebo (p < 0.01).By week 12, the Rz index had decreased 19.7% vs. placebo (p < 0.01).

Expert Visual Grading and Self-Perception of AC Efcacy for Skin Appearance.
As can be seen in Table 1, after 12 weeks of daily use of AC supplement, the Glogau Score had improved by 8.54% compared to placebo (p < 0.01).VAS score for expert grading of skin tone evenness showed Dermatology Research and Practice a 31.9%improvement in skin tone vs. placebo (p < 0.01).By week 12, 87.5% of subjects reported that their skin felt more hydrated (vs.63.4% in the placebo group), 72.9% of subjects reported their skin felt frmer (vs.50% in the placebo group), 62.5% reported that their wrinkles appeared reduced (vs.36.5% in the placebo group), and 62.5% of subject reported their skin had more elasticity and felt stronger from within (vs.28.8% in the placebo group).SPQ responses were statistically signifcant (p < 0.05) except for the self-perception of wrinkle reduction.

Clinical Trichoscopy, Visual Grading, and SPQ Show AC
Use Improves Scalp and Hair Condition.A trichoscope camera system was used to magnify scalp surface features and hair follicles up to 60-fold.Te scalp was assessed for infammation, faking, plugging, and scaling.When using the AC supplement daily for 12 weeks, we measured an average increase of 11 ± 29% improvement in VAS scalp scaling vs placebo (no change).Tis corresponded to an average increase of 33.4 ± 26.2 total hairs counted per unit area (representing a 27.6% increase vs. placebo, p � n.s.).A detailed clinical analysis of scalp sebum, follicular plugging, and infammation, in parallel to total hairs per follicular unit and mean hair diameter, shows improvements when using the AC supplement daily (manuscript in preparation for trichology publication).By expert visual grading, hair healthy appearance was assessed on a 5-point scale.VAS score for hair healthy appearance improved 31.9% for subjects taking the AC supplement daily vs. 9.4% of subjects who took placebo daily (p < 0.01).By week 12, SPQ reported that 66.7% of subjects felt their hair thicker vs. 44.2% in the placebo group (p � 0.02), whereas 77.1% of subjects reported that their hair felt smoother vs. 55.8% in the placebo group (p < 0.01).

AC Use Daily Shows Superior Efcacy Compared to Use
Every 48 hours.As can be seen in Table 2 and Figure 5, for subjects consuming the AC supplement daily, by week 12, the fragmentation VAS grade had decreased 44.6% compared to subjects taking the supplement on every second day, in which case fragmentation grades had decreased 10.6%.Daily use of the AC supplement showed signifcantly improved collagen density, integrity, and a reduction of fragmentation compared to use every 48 hours (p < 0.01); that is, the diference between the 2 treatment regimens was statistically signifcant.
For subjects taking the AC supplement daily, by week 12, the elasticity index, R2, had increased 22.7% compared to subjects taking the supplement on every second day, in which the R2 index had increased 18.6%.Te diference between the 2 treatment regimens was not statistically signifcant.By week 12, for subjects taking the AC  Dermatology Research and Practice supplement daily, the Rz index had decreased 19.7% compared to the group taking the supplement on every second day, in which the Rz index had decreased 12.5%, with the diference being statistically signifcant (p < 0.01).Expert grading VAS score for skin tone evenness showed a 31.9%improvement at week 12 compared to the group taking the supplement on every second day, which improved 26.3%, with the diference being statistically signifcant (p � 0.03).

Exploratory Postsupplementation
Phase.After the 12week clinical trial ended, subjects discontinued the use of the AC supplement.A subset of 32 participants returned after another 8 weeks of additional skin measures (hydration, elasticity, and and self-perception feedback.Furthermore, the data for week 12 are diferent for the full clinical study because we only compare the scores for the 32 participants of this exploratory phase of the RCT (as opposed to the full clinical study which tested 48 participants).For comparative purposes, data for hydration, R2 and Rz were normalised by expressing as percent change from baseline.Each parameter was expressed relative to placebo.With daily use of the AC supplement, hydration and elasticity showed an increase (i.e., an improvement in skin quality) both at week 6 and week 12 compared to placebo (p < 0.01).Wrinkle size, Rz, was reduced (i.e., less visible in size) both at week 6 and week 12 compared to placebo (p < 0.01) ( * * denotes p < 0.01).After taking AC supplement for 12 weeks, hydration had increased by 11%, but when the participants stopped taking AC at week 20, it had decreased by 4%.Elasticity R2 index had increased by 53% at week 12, but at week 20, the R2 index dropped to only 31%.Wrinkles had decreased by 20% at week 12, whereas at week 20, the Rz index had dropped to 8%.Tis indicates that when subjects cease using the supplement, then there is a measurable loss of skin benefts.Tis exploratory part of the study will be used for a statistical power analysis to estimate the minimum sample size required for a follow-up clinical trial.

Discussion
Photodamage is characterised by many attributes including leathery texture, thin, fattened and dry features, fne lines and wrinkle formation, poor elastic recoil, fragility of the skin, and altered pigmentation due to haemoglobin and melanin [18].For over a decade, a key hypothesis proposes that many of these problems are putatively related to loss of collagen, which can be reversed by the ingestion of hydrolysed collagen supplements [19].
In this randomised, double-blind, placebo-controlled clinical trial, we have provided evidence linking the use of hydrolysed collagen supplements, with increased collagen content and quality in the skin.We demonstrated that this increased collagen in the skin is associated with improved appearance indices such as hydration, elasticity, and wrinkles.Te study was run during Q1, 2023 to efectively minimise variability due to seasonal changes, especially increased UV-radiation during summer and anticipated damage caused by UV-radiation to both dermal (collagen fragmentation and wrinkle formation) and epidermal (hydration and pigmentation) features.
Collagen is a uniquely stable and long-lived protein with a half-life reported to be approximately 15 years [20,21].Over that duration of time, it can be infuenced by extrinsic and intrinsic factors, including UV-radiation, glycation, hormonal fux, oxidative stress linked to freeradical attack, mechanical shear stress, and physical fatigue.In addition to direct damage, the production of matrix metalloproteinase (MMP) enzymes, in particular collagenase, can cleave the collagen triple helix and make the fbre accessible to degradation enzymes and cellular recycling [22].Te collagenases are critical to carry out the frst degradation step, in which the fbres are cleaved into characteristic 1/4 and 3/4 fragments [11,23].Te resultant fragments can be visualised, quantifed, and represent collagen damage in the dermis.
Te collagen fbres in situ can be described as hyporefective (where it is not possible to identify single fbres), hyperefective (in which a well-defned, fbrous collagen can be observed), thin reticulated collagen, coarse collagen, huddle collagen, and curled bright structures [24].According to Bhardwaj et al., short fragmented collagen and a huddled arrangement are typically seen in cases of damaged and poorly oriented collagen in aged or photodamaged skin [25].
Both the amount and quality of the collagen network changes over time, with state-of-the-art confocal laser scanning microscopy (CLSM) being used to visualise and quantify such changes.We have presented images of the dermis in the crow's  Dermatology Research and Practice feet area of the face at baseline and 12 weeks after the oral intake of AC supplement vs. placebo.At baseline, there is low collagen density observed and a lack of structural fbres which usually represent collagen-based ECM.Tis corresponds to visible evidence of fragmented collagen which has been reported to underline loss of skin elasticity and appearance of facial wrinkles.Following daily use of the AC supplement, an increase in collagen density and structural integrity can be observed in parallel to a decrease in fragmentation.Tis improvement in dermal collagen content was not observed in the placebo group.Tis is in agreement with week 12 results presented by Asserin et al. [17], albeit we have shown the efect occurring as early as week 6, with progressive improvements over the 12-week period.We have also shown, for the frst time, that this beneft is dependent on the dosing regimen.
In addition to fragmentation, it is important to consider the density and quality of collagen in the dermis, paying particular attention to the stratifcation of collagen in relation to papillary and reticular fbroblast populations [26,27].Papillary fbroblasts in the upper dermis have been reported to secrete distinctly discernible thin, poorly organized bundled collagen fbres, compared to the reticular fbroblasts which secrete thick, well-organized collagen bundles.Tere is also a distinct pattern of the papillary ECM exhibiting a higher ratio of collagen type III to type I, a pattern often observed in younger skin and also in wound healing responses [11,28].
Te characteristic organized, parallel-oriented, and bundled collagen fbrils are one of the key echogenic structures of the dermis.Te ultrasonographic markers included % pixel area (corresponding to collagen density), LEP, MEP, HEP, and the ratio of LEP u /LEP i .Te LEP indicator quantifes the degree of cutaneous hydration, infammatory processes, solar elastosis, and collagen degeneration.Te MEP and HEP indices quantify the structures of collagen and elastin fbres and microfbrils.In general, an increase in collagen density, MEP, and HEP was observed (all which correspond to increased collagen in the dermis), although only the HEP showed an improvement compared to placebo.Te variability in echogenic markers and strong placebo efect we hypothesise is due to the superimposition of innate collagen turnover mechanisms in combination with efects of the supplement.
Te increase in HEP pixel data is indicative of increased collagen content and quality in the dermis.When considered in the context of a decrease in the LEP u /LEP i ratio, we surmise that this is evidence of improvements in the papillary and upper dermis.Tis is in agreement with previous publications on changes on the LEP u /LEP i ratio in the aged skin and following treatment with antiaging topical treatments (such as the topical favonoid and ε-viniferin) but needs to be independently confrmed in the case of daily use of HC supplements [29][30][31].
Many of the published antiaging, hydrolysed collagen clinical trials, do not add vitamin C to the product being tested.We regard the inclusion of vitamin C as a key factor driving efcacy of these supplements.Te role of vitamin C (l-ascorbic acid) as a cofactor required in the synthesis of collagen and the ECM is well documented, but the importance of this vitamin as an antioxidant is often overlooked.Free radical damage to the skin is triggered by several mechanisms including UV-radiation, mitochondrial oxidative respiration, glycation, and lipid oxidation.Ascorbic acid neutralises a diverse number of free-radical species to facilitate their detoxifcation [32].Te consequent generation of a stable ascorbyl free radical yields a far less reactive species which efectively minimises free-radical damage and protects the long-lived collagen fbres in the skin [33].
Linking changes in dermal collagen content to phenotypic and visible skin attributes is important to the overall acceptance and credibility of the use of hydrolysed collagenbased supplements.Studies using proflometry show the architecture of the uppermost layers of the skin, either as microrelief (including fnes lines) or larger structures such as wrinkles.Te skin microrelief changes with age independently of wrinkle formation, and thus any model of proflometric analysis of the skin needs to account for these independent characteristics.Fine lines and wrinkles were signifcantly decreased at both timepoints, and this improvement was not observed in the placebo group.
Skin hydration and elasticity were signifcantly increased after 6 weeks and 12 weeks use of the AC supplement, in direct contrast to the placebo which showed negligible change from baseline, or even a deterioration of skin parameters at both timepoints.Te efects of supplement on elasticity are likely due to changes in collagen content in addition to changes in elastin and glycosaminoglycan content of the dermis.Te decrease in hydration observed in the placebo group is likely due to the fact that the clinical trial was run during the winter season, where many people experience some degree of dry skin/winter xerosis, associated with low temperature and low humidity.
Te use of supplements as a support to a healthy and varied diet is an issue of popular debate.A key question arises as to the frequency of use of the supplement and specifcally whether daily use of the will yield more, or less, skin benefts compared to less frequent use (e.g., every 48 hours).We tested this hypothesis in a parallel cohort to the main clinical evaluation group.Te statistical model used to evaluate these data is referred to as the ordinary least squares regression model.Tis model is not biased by the fact that the two groups, of daily and 48 hour supplement use, have diferent number of subjects (n � 100 vs. n � 30, respectively).If each group has a sufcient number of subjects to determine signifcance, the overall model will be sufciently powerful in determining the signifcance between groups, thus minimising the possibility of statistical bias when comparing daily use of the supplement, with use every 48 hours.
Te choice to take supplements daily, or on alternate days, can be driven by information derived from online content, by personal preferences, or by fnancial constraints.Regardless of a reason, there was to date no published evidence to support the impact of the dosing regimen on clinical efcacy and benefts when taking hydrolysed collagen supplements.We present here the frst clinical evidence, measured against placebo, that proves that taking 10 Dermatology Research and Practice a supplement on alternate days will decrease the benefts and reduce efcacy for skin collagen, wrinkles, and skin tone.We have shown that taking the AC supplement daily is more efcacious than use every 48 hours, and this should be borne in mind by consumers who wish to vary their dosing regimen.

Conclusion
We have shown that an AC supplement, when consumed daily over 12 weeks, can improve collagen content in the dermis.Furthermore, there is an association between collagen fbres in the dermis and aging features such as hydration, elasticity, and wrinkles.We have extended this to include trichoscope and clinical assessment of scalp and hair condition.We have shown that the efcacy of the supplement is directly dependent on dose regimen, with subjects who take the supplement every 48 hours show less efcacy than subjects who take the AC supplement daily.Tese data clearly demonstrate that taking a marine-sourced, hydrolysed collagen plus vitamin C supplement daily can improve skin, scalp, and hair.

Figure 1 :
Figure 1: Clinical fowchart.Trial design, including subject allocation and participation over 12 weeks.

Figure 3 :
Figure 3: Efect of supplement on the collagen density, structure, and fragmentation.Representative images from 1 subject in the treatment group and the placebo group are shown.Confocal images show low collagen density, poor structural integrity of fbres and a visible fragmentation at baseline for both groups.At week 12 there is an increase in collagen density, increased presence of fbrillar structures and a decrease in the fragmentation pattern for subjects using the AC supplement daily.

Figure 4 :
Figure4: Summary of skin parameters for hydration, elasticity, and wrinkles.For comparative purposes, data for hydration, R2 and Rz were normalised by expressing as percent change from baseline.Each parameter was expressed relative to placebo.With daily use of the AC supplement, hydration and elasticity showed an increase (i.e., an improvement in skin quality) both at week 6 and week 12 compared to placebo (p < 0.01).Wrinkle size, Rz, was reduced (i.e., less visible in size) both at week 6 and week 12 compared to placebo (p < 0.01) ( * * denotes p < 0.01).

Figure 5 :
Figure 5: Summary of AC use daily vs. use every 48 hours.For comparative purposes, data for collagen density and fragmentation, the elasticity R2 index, proflometry Rz index (wrinkles), and expert visual grading of skin even tone (pigmentation distribution) are expressed as percent change from baseline.Each parameter was expressed as daily use of the supplement or use every 48 hours ( * p < 0.05; * * p < 0.01).

Table 1 :
Summary for the skin biophysical and clinical assessments.Descriptive and probability statistics for the skin biophysical parameters and expert visual grading scales are presented.Te table shows the mean and standard deviation for each measurement, with statistical signifcance for change from baseline, and for treatment vs placebo (at Week 6 and atWeek 12).A threshold of p ≤ 0.05 was applied in all cases.

Table 2 :
Daily use of AC supplement vs. use every 48 hours.Descriptive and probability statistics are presented.Te table shows the mean and standard deviation at baseline and week 12, and percent change from baseline for each measurement at week 12. Statistical signifcance of the comparison of supplement use daily vs. use every 48 hours is presented.A threshold of p ≤ 0.05 was applied in all cases.